Test 3 Concepts

Cytoskeleton and Vesicle Trafficking

What is the cytoskeleton, what is its characteristics, and what are some of its functions

• The skeleton of the cell

• Network of filaments and tubules in cytosol that provides a structural scaffolding or framework for the cell

  • Monomers polymerize to form long filaments or tubular structures

• It may be very rigid, yet it is very dynamic: constantly changing in most cells

• Functions:

  • Defines cell shape

  • Supports the plasma membrane

  • Vesicle movement and organelle localization

  • Cell movement

  • Cell division

  • Cell-extracellular matrix interactions

What are the 3 components of the cytoskeleton and what are their sizes

• Microtubules

  • Subunit: α-β-tubulin heterodimers

  • Largest part (most wide) of the cytoskeleton

• Microfilaments

  • Subunit: G-actin monomers

  • Smallest part (most narrow) of the cytoskeleton

• Intermediate Filaments

  • Intermediate in size

  • Subunit: IF dimer

  • Variety of proteins that compose intermediate filaments

What has been discovered by studying microtubule polymerization in vitro (in a lab)

• α & β tubulin dimers polymerize to form microtubules

• Rate of polymerization depends on the concentration of tubulin dimer

  • At a high dimer concentration microtubules will polymerize

  • At a low dimer concentration microtubules will depolymerize

    • Results in free dimers that can be polymerized again

    • Causes pattern of shrinking and regrowing

• Tubulin dimers are asymmetrical, thus microtubules are asymmetrical

  • One end looks different than the other end

    • β tubulin is exposed at one end (plus + end)

    • α tubulin is exposed at the other end (minus - end)

How does GTP contribute to the growth of microtubules

• Both α and β tubulin bind to a molecule of GTP for the dimer to polymerize and join the growing microtubule

• Shortly after the dimer joins the microtubule, the GTP bound to β tubulin will be hydrolyzed

• When the dimer concentration is high there will be rapid growth and the
  microtubule has a GTP cap

  • Facilitates the continued addition of tubulin

  • GTP cap is stable and is a good binding site for more tubulin dimer

  • Leads to rapid rate to microtubule growth

What is microtubule catastrophe

When the dimer concentration falls too low and rate of addition of tubulin dimer slows down, polymerization ceases

• Lower dimer binding allows β tubulin to start hydrolyzing their GTP to GDP

• The rate GTP hydrolysis > rate of polymerization

  • GTP cap is lost

  • Loss of GTP cap makes the microtubule unstable

• GDP bound dimers have lower binding affinity for each other and the microtubule becomes unstable

• Depolymerization > polymerization = “microtubule catastrophe”

What is dynamic instability of microtubules

Rapid degradation or growth of microtubules

• Catastrophe can be followed by rescue of the microtubule as it alternates between growth and shrinkage based on the concentration of tubulin dimer

What is microtubule nucleation in vivo (in nature)

The beginning of the process of creating microtubules in cells

• Start with individual dimers and put them together to start making microtubules

• Microtubule nucleation requires γ-tubulin

• 13 copies of γ-tubulin (shown in green) are organized with other proteins into a structure called the γ-tubulin-ring complex

  • Serves as the anchor point for the minus (-) end of microtubules

  • γ-tubulin-ring complexes are found in microtubule organizing centers

    • The site where microtubules are nucleated (originate) and extend in all directions

    • Centrosome = where most MTOC is found

What are microtubule organizing centers and where are they mainly found

The site where microtubules are nucleated (originate) and extend in all directions

• In most cells, the primary MTOC is located in the centrosome

  • The centrosome contains pair of centrioles + numerous additional proteins that compose the pericentriolar material

    • Peri = around, stuff around the centriole

    • We don’t know what centrioles do; plants have microtubules but no centrioles

    • Believed that centrioles to contribute to establishing the centrosome

Other MTOC locations

• Epithelial cells that form cilia

  • Basil bodies structure is identical to a single centriole

  • Basil bodies help form cilia

• Nerve cells

• Red blood cells

• Centrosomes in cell division

Fluorescence microscopy

• Microtubules in yellow

• MTOC behind the nucleus (bright light in center)

• DNA in magenta

• Microfilaments in blue

Microtubule toxins

• Inhibit polymerization: colchicine, nocodazole, vincristine

  • Most work by binding up the dimer and preventing the dimer from being added to the microtubule

  • Used in chemotherapy for cancer treatment

  • Pulse/chase experiment with colcemid

• Inhibit depolymerization: paclitaxel

  • Stabilizes intact microtubules so that they can never shrink

  • Also used in chemotherapy

How are microtubule toxins used in cancer treatment

• Toxin that inhibit polymerization (colchicine, nocodazole, vincristine):

  • Prevent microtubules from forming so that mitosis will not occur

• Toxins that inhibit depolymerization (paclitaxel):

  • Prevents mitosis from continuing because microtubules cannot shrink back and pull DNA to opposite ends of the cell

How are microtubule functions regulated in vivo

Regulated by microtubule associated proteins (MAPs)

• MAPs either interact with tubular dimer or intact microtubules

• Two classes:

  • Motor MAPS: kinesin & dynein

  • Non-motor MAPS

What are non-motor MAPs and how do they regulate microtubule function

• A class of microtubule associated proteins (MAPs)

• Numerous proteins bind to the plus end of microtubules (+TIPs) and promote:

  • Polymerization

  • Stabilization

  • Depolymerization (promote catastrophe)

• Some proteins bind laterally to microtubules and promote crosslinking: tau, MAP2 (spacing between microtubules is much larger)

What are the two non-motor MPAs that bind laterally to the microtubules and what is the difference between them

tau and MAP2

• MAP2 has greater spacing between the microtubules

How are microfilaments formed and what is its structure

Monomers of globular protein called actin (G-actin) polymerize to form filamentous actin (F-actin or microfilaments)

• Rate of polymerization depends on the concentration of G-actin

• G-actin is asymmetrical, thus F-actin is asymmetrical (+ end & - end)

• G-actin must be bound to ATP in order to be added to the growing microfilament

• Shortly after G-actin addition, the ATP bound to it will be hydrolyzed

What are the actin binding proteins and how do they bind to actin to affect microfilament structure (provide examples)

• Proteins that bind to actin to affect microfilament structure and structure

• Some bind G-actin and regulate:

  • Polymerization: formin & profilin

  • Depolymerization: cofilin

• Some bind F-actin and regulate:

  • Filament length:

    • CapZ

      • Binding on the end and preventing any more filament addition

  
  

  • Formation of networks

    • filamin

      • Bind to filament and creates cross-linked structures of filaments

      • As filaments cross, they are linked together

      • Can cause gel-like consistency of cytosol; becomes thicker

  
  

  • Severing F-actin:

    • gelsolin

      • Cuts crosslinked filaments into fragments and caps them so they don’t polymerize and grow anymore

      • Cytosol becomes solvent-like

  
  

  • Branching:

    • Arp2/3

      • Allow microfilaments to create a branch point and creates branching filaments

  
  

  • Formation of bundles:

    • fimbrin, α-actinin

      • Facilitates parallel formation of bundles

What structure do specialized actin assemblies create and how

• Microvilli

• Parallel bundles of microfilament that push the membrane out

• Common in epithelial cells

• Increases the surface area of digestive system cells for transport

How do actin assemblies help with cell motility

Fibroblasts, immune system cells, neutrophils, macrophages, etc.

• Filopodium on a leading edge

  • Parallel bundle structures

• Lamellipodium

  • branched network pushes the whole membrane forward

• Cell cortex

  • in the gel, gives stability for filopodium and lamellipodium

• Stress fiber

  • in contractile bundle that retracts the trailing edge of the cell

  • Antiparallel structure

How are actin networks formed

Arp2/3

• A dimer called actin-related protein

• Binds laterally along microfilaments

• nucleates branch points on existing filaments

How are the proteins that form actin networks regulated

The Rho family of small G-proteins regulate the formation of different
actin assemblies by regulating proteins the actin binding proteins

• Rho, Rac, Cdc42

How do Rho family members contribute to the creation of actin assemblies used in cell motility

Overexpression of ‘dominant active’ forms of each G-protein

• Unable to hydrolyze their GTP

• Normal cell on the very left

• Rho creates stress fibers

• Rac creates Lamellipodium

• Cdc42 creates Fliopodium

What are some proteins that make intermediate filaments

• Lamin proteins provide structure support to the nuclear membrane

• Keratin